Genome-Wide Identification of Regulatory Elements and Reconstruction of Gene Regulatory Networks of the Green Alga <i>Chlamydomonas reinhardtii</i> under Carbon Deprivation

<div><p>The unicellular green alga <i>Chlamydomonas reinhardtii</i> is a long-established model organism for studies on photosynthesis and carbon metabolism-related physiology. Under conditions of air-level carbon dioxide concentration [CO₂], a carbon concentrating mechanism (CCM) is induced to facilitate cellular carbon uptake. CCM increases the availability of carbon dioxide at the site of cellular carbon fixation. To improve our understanding of the transcriptional control of the CCM, we employed FAIRE-seq (formaldehyde-assisted Isolation of Regulatory Elements, followed by deep sequencing) to determine nucleosome-depleted chromatin regions of algal cells subjected to carbon deprivation. Our FAIRE data recapitulated the positions of known regulatory elements in the promoter of the <i>periplasmic carbonic anhydrase</i> (<i>Cah1</i>) gene, which is upregulated during CCM induction, and revealed new candidate regulatory elements at a genome-wide scale. In addition, time series expression patterns of 130 transcription factor (TF) and transcription regulator (TR) genes were obtained for cells cultured under photoautotrophic condition and subjected to a shift from high to low [CO₂]. Groups of co-expressed genes were identified and a putative directed gene-regulatory network underlying the CCM was reconstructed from the gene expression data using the recently developed IOTA (inner composition alignment) method. Among the candidate regulatory genes, two members of the MYB-related TF family, <i>Lcr1</i> (<i>Low-<u>C</u>O</i>_<i>2</i><i> response regulator 1</i>) and <i>Lcr2</i> (<i>Low-<u>C</u>O</i>_<i>2</i><i> response regulator 2</i>), may play an important role in down-regulating the expression of a particular set of TF and TR genes in response to low [CO₂]. The results obtained provide new insights into the transcriptional control of the CCM and revealed more than 60 new candidate regulatory genes. Deep sequencing of nucleosome-depleted genomic regions indicated the presence of new, previously unknown regulatory elements in the <i>C. reinhardtii</i> genome. Our work can serve as a basis for future functional studies of transcriptional regulator genes and genomic regulatory elements in <i>Chlamydomonas</i>.</p> </div

Comparison of enrichment of regulatory elements at the <i>Cah1</i> locus.

<p>Quantitative PCR analysis of FAIRE fragments was performed for the <i>Cah1</i> locus, with primers designed for covering coding and non-coding regions, represented by the amplicon identifier (Table 1). Enrichment of FAIRE fragments within the <i>Cah1</i> locus was analyzed by comparing the chromatin from cells cultured under HC and LC condition. Enrichment observed for the 5´ upstream and 3´ downstream regions of the gene locus is more pronounced in cells cultivated at LC, indicating that these regions may have regulatory functions. </p

Cumulative histogram of the approximate distances of FAIRE summits from their next closest annotated gene.

<p>Genomic coordinates were used to calculate the distances between FAIRE summits and the start and stop codons of their nearest gene transcripts. The number of transcripts with at least one FAIRE peak assigned was counted and the distances to the start or stop codons computed. The gray line indicates the cumulative percentage of the transcripts to which FAIRE peaks were assigned. As seen, approximately 80% of the FAIRE summits were located within 1.5 kb up- or downstream of the transcript´s start or stop codon. FAIRE peaks were detected using MACS tool v. 1.4.0beta, with a <i>p</i>-value cutoff equal to 10^-05.</p

Quantitative PCR of FAIRE fragments derived from the <i>Cah1</i> locus.

<p>(A) Quantitative PCR analysis was performed for ten DNA segments of the <i>Cah1</i> locus and enrichment of each fragment was measured. Red areas show the location of the DNA segments amplified and quantified. Exons are indicated by blue boxes and introns by dashed lines. (B) DNA segments located in the promoter region (amplicons B and C) and in a region located in the last exon of the <i>Cah1</i> gene, representing the 3´ UTR (amplicons I and J), were found to be enriched. De-crosslinked chromatin was used as control for calculating the enrichment of genomic fragments in the FAIRE samples. The enrichment was determined as 2^-ΔΔCt (cf. Material and Methods) and error bars indicate the standard deviation (SD) from three biological replicates. The reference region H is indicated by a star (*) in (A) and refers to the region where the C_t value ratio between FAIRE and control samples was the lowest. The C_t values of the reference region were used for the calculation of the FAIRE enrichment as a normalization parameter. The position of regulatory elements of the LC response previously identified by Kucho et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079909#B24" target="_blank">24</a>] is indicated. </p

Web FAIRE-seq GenomeBrowser.

<p>FAIRE-seq reads mapped to the <i>Chlamydomonas</i> genome annotation v.4 were made accessible through a web GenomeBrowser (<a href="http://bce.uniandes.edu.co/gb2/gbrowse/chlamydomonas_v4/" target="_blank">http://bce.uniandes.edu.co/gb2/gbrowse/Chlamydomonas_v4/</a>). (A) <i>Cah1</i> locus. The annotated <i>Cah1</i> transcript is included; the yellow horizontal boxes indicate annotated exons and the connecting black lines indicate introns. Positions of FAIRE peaks are indicated as green horizontal bars, FAIRE summits as vertical red bars. Sequence coverage along the <i>Cah1</i> locus is indicated by the gray vertical bars; the darkness of the gray bars is proportional to the abundance of the sequencing reads mapped to this particular location. (B) Broader view of the genomic location where <i>Cah1</i> is annotated. Other transcripts in the proximity of the <i>Cah1</i> locus are shown, together with their corresponding features.</p

TF and TR genes co-expressed under conditions of low CO₂ concentration.

<p>The analysis of the TF and TR transcript levels revealed co-expressed genes, clustered according to similar expression profiles using the quality threshold method with Pearson correlation as similarity measure. The y axis shows normalized expression levels as relative expression values (log₂), scaled by gene minima and maxima and mean centered. At time zero (0 min) the carbon dioxide concentration in the growth medium was shifted from 5% to 0.04%. Samples were taken immediately before the shift (indicated as 0 min) and after 60, 120 and 180 min. Sampling time points are shown on the x axis.</p

Subcutaneous anti-COVID-19 hyperimmune immunoglobulin for prevention of disease in asymptomatic individuals with SARS-CoV-2 infection: a double-blind, placebo-controlled, randomised clinical trialResearch in context

Summary: Background: Anti-COVID-19 hyperimmune immunoglobulin (hIG) can provide standardized and controlled antibody content. Data from controlled clinical trials using hIG for the prevention or treatment of COVID-19 outpatients have not been reported. We assessed the safety and efficacy of subcutaneous anti-COVID-19 hyperimmune immunoglobulin 20% (C19-IG20%) compared to placebo in preventing development of symptomatic COVID-19 in asymptomatic individuals with SARS-CoV-2 infection. Methods: We did a multicentre, randomized, double-blind, placebo-controlled trial, in asymptomatic unvaccinated adults (≥18 years of age) with confirmed SARS-CoV-2 infection within 5 days between April 28 and December 27, 2021. Participants were randomly assigned (1:1:1) to receive a blinded subcutaneous infusion of 10 mL with 1 g or 2 g of C19-IG20%, or an equivalent volume of saline as placebo. The primary endpoint was the proportion of participants who remained asymptomatic through day 14 after infusion. Secondary endpoints included the proportion of individuals who required oxygen supplementation, any medically attended visit, hospitalisation, or ICU, and viral load reduction and viral clearance in nasopharyngeal swabs. Safety was assessed as the proportion of patients with adverse events. The trial was terminated early due to a lack of potential benefit in the target population in a planned interim analysis conducted in December 2021. ClinicalTrials.gov registry: NCT04847141. Findings: 461 individuals (mean age 39.6 years [SD 12.8]) were randomized and received the intervention within a mean of 3.1 (SD 1.27) days from a positive SARS-CoV-2 test. In the prespecified modified intention-to-treat analysis that included only participants who received a subcutaneous infusion, the primary outcome occurred in 59.9% (91/152) of participants receiving 1 g C19-IG20%, 64.7% (99/153) receiving 2 g, and 63.5% (99/156) receiving placebo (difference in proportions 1 g C19-IG20% vs. placebo, −3.6%; 95% CI -14.6% to 7.3%, p = 0.53; 2 g C19-IG20% vs placebo, 1.1%; −9.6% to 11.9%, p = 0.85). None of the secondary clinical efficacy endpoints or virological endpoints were significantly different between study groups. Adverse event rate was similar between groups, and no severe or life-threatening adverse events related to investigational product infusion were reported. Interpretation: Our findings suggested that administration of subcutaneous human hyperimmune immunoglobulin C19-IG20% to asymptomatic individuals with SARS-CoV-2 infection was safe but did not prevent development of symptomatic COVID-19. Funding: Grifols